Mechanisms of Wharton’s Jelly-derived MSCs in enhancing peripheral nerve regeneration

Warton’s jelly-derived Mesenchymal stem cells (WJ-MSCs) play key roles in improving nerve regeneration in acellular nerve grafts (ANGs); however, the mechanism of WJ-MSCs-related nerve regeneration remains unclear. This study investigated how WJ-MSCs contribute to peripheral nerve regeneration by examining immunomodulatory and paracrine effects, and differentiation potential. To this end, WJ-MSCs were isolated from umbilical cords, and ANGs (control) or WJ-MSCs-loaded ANGs (WJ-MSCs group) were transplanted in injury animal model. Functional recovery was evaluated by ankle angle and tetanic force measurements up to 16 weeks post-surgery. Tissue biopsies at 3, 7, and 14 days post-transplantation were used to analyze macrophage markers and interleukin (IL) levels, paracrine effects, and MSC differentiation potential by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The WJ-MSCs group showed significantly higher ankle angle at 4 weeks and higher isometric tetanic force at 16 weeks, and increased expression of CD206 and IL10 at 7 or 14 days than the control group. Increased levels of neurotrophic and vascular growth factors were observed at 14 days. The WJ-MSCs group showed higher expression levels of S100β; however, the co-staining of human nuclei was faint. This study demonstrates that WJ-MSCs' immunomodulation and paracrine actions contribute to peripheral nerve regeneration more than their differentiation potential.


Expression of macrophage markers
The mRNA expression levels of CD206 and IL10 were not significantly different between the two groups at 3 days postoperatively.The mRNA expression level of CD206 was significantly higher in the WJ-MSCs group than in the control group at 14 days postoperatively (p < 0.05).Meanwhile, the mRNA expression level of IL10 was significantly higher in the WJ-MSCs group than in the control group at 7 and 14 days postoperatively (p < 0.05) (Fig. 2A).In the analysis of protein expression levels, CD206 expression was maintained at a higher level in the WJ -MSCs group than in the control group until 14 days postoperatively.However, CD68 expression was not different between the two groups (Fig. 2B).

Expression of neurotrophic and angiogenesis factors
The mRNA expression levels of the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factors (VEGF) were not significantly different between the two groups at 3 and 7 days postoperatively.However, the mRNA expression levels of BDNF and VEGF were significantly higher in the WJ-MSCs group than in the control group at 14 days postoperatively (p < 0.05) (Fig. 3A).In addition, immunofluorescence staining confirmed that the protein levels of NGF, BDNF, and VEGF were higher in the WJ-MSCs group than in the control group, showing a similar pattern to RNA expression (Fig. 3B).

Expression of SC markers and results of double-staining with human nuclei and S100β
The mRNA expression levels of S100β and myelin basic protein (MBP) were not significantly different between the two groups at 3 days postoperatively.However, they were significantly higher in the WJ-MSCs group than in the control group at 7 days postoperatively (p < 0.05).This difference was maintained until 14 days postoperatively only for MBP (p < 0.05).In addition, immunofluorescence staining showed similar results (Fig. 4).Double-staining of human nuclei and S100β in the WJ-MSCs group showed that the red-stained human nuclei and green-stained S100β did not co-colonize at 7 days postoperatively (Fig. 5).

Discussion
Over the years, MSCs have attracted considerable interest in regenerative medicine; they are being considered in in vitro and in vivo studies to test their efficacy in enhancing peripheral nerve regeneration 16 .Additionally, they are believed to replace injured tissue cells via differentiation and maximize the intrinsic regenerative capacity of injured tissues by producing several growth factors and cytokines 17 .The mechanisms of MSCs for enhancing nerve regeneration depend on their type; however, no in-depth studies have evaluated these mechanisms in WJ-MSCs 18 .
After peripheral nerve injury, SCs trigger a series of immunoregulatory reactions by secreting cytokines and recruiting various immune cells 19 .In this process, MSCs can produce various immunoregulatory factors that modulate immune function (immunomodulatory effects) 20 .This phenomenon has been revealed in several types of MSCs 21 , but not in WJ-MSCs.Generally, macrophages can be divided into two groups: M1 macrophages, which stimulate local inflammation and remove debris at the injury site, and M2 macrophages, which play an important role in wound healing and tissue repair 22 .In this study, the WJ-MSCs group showed significantly higher level of macrophage markers, including CD206, which is a marker for an anti-inflammatory M2 macrophage 23 and IL10, which is known for its anti-inflammatory properties 24 .However, the protein expression level of CD68, as a panmacrophage marker that reflects macrophage quantities of M1 and M2 25,26 , was not significantly different between the two groups.This finding indicates that the number of recruited macrophages was not significantly different.The roles of M1 and M2 macrophages are nuanced and flexible 27 .Pro-inflammatory macrophages can even convert to anti-inflammatory phenotypes, demonstrating their remarkable plasticity 28 .WJ-MSCs can enhance the polarization of macrophages toward the M2 phenotype, promoting an anti-inflammatory environment 29 .Antiinflammatory factors have several beneficial effects on nerve regeneration, including activating and proliferating SCs 30 , promoting angiogenesis 31 , and modulating the ECM environment to favor axonal growth 32 .
It is known that MSCs secrete various factors that promote nerve cell survival and axonal growth, showing increased paracrine effects 18,33 .In this study, the WJ-MSCs group showed significantly higher levels of neurotrophic (BDNF and NGF) and angiogenesis factors (VEGF).Additionally, BDNF is a neurotrophic factor that supports neuronal survival, growth, and differentiation 34 .After peripheral nerve injury, BDNF is upregulated and sustained over the course of weeks 35,36 .It promotes axonal growth, provides essential cues for axonal sprouting, and directs axonal regeneration toward their target, facilitating functional recovery 37,38 .Similar to BDNF, NGF stimulate neuronal survival, promote axonal growth, and exerts a neuroprotective effect against nerve system disorders 39,40 .Further, VEGF is a potent angiogenic factor that plays a critical role in angiogenesis, which is essential for the revascularization of regenerating nerves 41 .It exhibits direct neuroprotective effects on neurons 42 and enhances SC proliferation and migration after peripheral nerve injury 43 .In addition to BDNF and VEGF, WJ-MSCs secrete various growth factors and cytokines that collectively contribute to paracrine effects and promote nerve regeneration.These factors include NGF, fibroblast growth factor, and insulin-like growth factor 18 .
In this study, the WJ-MSCs group exhibited higher levels of SCs markers than the control group.To determine the reason for the higher levels of SCs markers (S100β) in the WJ-MSC group, we performed the double staining of human nuclei and the S100β antibodies.If the transplanted WJ-MSCs were differentiated into Schwann cells, the expression of S100β and h nuclei, as a specific marker of human cells, would have co-localized.However, double-staining of human nuclei and S100β in the WJ-MSCs group did not show co-colonization, suggesting that the observed increase in the SC markers may be attributed to the recruitment of host SCs rather than the direct differentiation of WJ-MSCs into SCs.WJ-MSCs can differentiate into SCLCs in vitro, which promote peripheral nerve regeneration in ANGs in vivo 5 .However, the differentiation of MSCs into SCLCs is a time-consuming process and typically requires the use of specific growth factors and signaling molecules 44 .To enhance peripheral nerve regeneration by utilizing the differentiation potential of MSCs, the in vitro application of differentiated MSCs may be more beneficial than using naïve MSCs 7 .Further studies are needed to confirm whether the benefits obtained from the direct differentiation of MSCs, when applied after differentiation, are greater than those obtained through the immunomodulatory and paracrine effects of naïve MSCs in peripheral nerve regeneration.
This study has several limitations.First, although we observed elevated levels of several markers in the WJ-MSCs group compared to the control group, we did not assess the specific biological role of each marker.Further experimental validation is necessary to confirm their effectiveness.Second, we were unable to discover new target gene and related to pathways using RNA-Seq.Discovering new marker and signaling, knocking out www.nature.com/scientificreports/or overexpressing them, and proving their relationship with peripheral nerve regeneration will lead to more innovative research.
In conclusion, the present study demonstrates that the mechanism of WJ-MSCs in enhancing peripheral nerve regeneration is because of their immunomodulatory and paracrine effects rather than their differentiation potential.This finding will be useful for understanding the mechanism of MSCs for enhancing peripheral nerve regeneration.

Preparation and culture of human WJ-MSCs
This study was approved by the Institutional Review Board of Asan Medical Center (No. 2015-0303), and the WJ-MSCs were provided by the Stem Cell Center, Asan Institute for Life Sciences, Seoul, Korea.All experiments were performed in accordance with relevant guidelines and regulations.Informed consent from the mothers was obtained for the use of umbilical cords.Umbilical cords were cut into 0.3-1.0cm pieces without blood vessels.The matrix was minced and transferred to culture dishes in minimal essential medium supplemented with 10% fetal bovine serum and an antibiotic-antimycotic mixture at 37 °C in a 5% CO 2 incubator in vitro as described previously 45 .When the cells reached 80% confluency, they were replated at a 1:3 split ratio.

Preparation of ANGs
This study complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Asan Medical Center and Ulsan University College of Medicine (No. 2017-12-127), and all the following methods were performed in accordance with the relevant guidelines and regulations.After isoflurane induction, rats were euthanized by CO 2 inhalation.Sciatic nerve segments (10 mm in length) were harvested from male Sprague-Dawley (SD) rats (7-8 weeks old, weight 250-350 g) (Orient Bio Inc., Seongnam, Korea).To prepare ANGs, sciatic nerve pieces were decellularized using a series of detergents as described by Shin et al. 2 .Briefly, the nerves were treated with detergents, including aprotinin, CHAPS, and DNase and RNase solutions.Then, the decellularized segments were washed several times with phosphate-buffered saline (PBS) to remove residual reagents and stored in PBS at 4 °C until use.All solutions were autoclaved or filter-sterilized before use.

Surgical procedure
Seventy-four adult male SD rats were randomly assigned to two groups: WJ-MSCs group (which was implanted with WJ-MSCs-laden ANG; n = 37) and control group (which was implanted with ANG only; n = 37).After anesthetization, the left sciatic nerve of rats was exposed and transected, and 10 mm of the nerve was removed.The 10-mm piece of WJ-MSCs-laden ANG or ANG was sutured using 9-0 nylon (Ethicon, Somerville, NY) under a microscope.

Functional assessment
Seven rats were selected from each group, and their ankle angles at the toe-off phase were measured at 4, 8, 12, and 16 weeks postoperatively to evaluate serial functional recovery.A walking track (length 1 m, width 10 cm, and height 10 cm) was built for this test.During the test, video was acquired with a digital camera (Canon SX730HS, Canon, Tokyo, Japan) at a distance of 1 m and calibrated to prevent optical distortion.Records were repeated until three satisfactory trials were obtained per rat.The ankle angle at the toe-off phase was measured at maximal plantar flexion in the experimental lateral ankle joint.After the foot and leg segments were manually identified in the video frames, the ankle angles at the toe-off phase were displayed in degrees.All rats were anesthetized at 16 weeks postoperatively, and the maximum isometric tetanic force was measured.The sciatic nerve was fully exposed through previous operation incision, and another skin incision was made anterior to the ankle to expose and transect the tibialis anterior (TA) tendon distally.The TA tendon was connected to a force transducer using a custom clamp with the knee and ankle joints immobilized to a platform.A bipolar stimulator (Grass S88, Grass Instrument Corp, Quincy, MA) was used to generate stimulus and processed on a computer using LabVIEW software (National Instruments, Austin, TX).All contractions were performed at supramaximal voltage to ensure maximal activation of all TA motor units.The strength of muscles was standardized as a percentage of the value from the contralateral side.

Figure 1 .
Figure 1.In vivo functional recovery.(A) Video analysis of gait angles at the toe-off phase.The angles were measured in the control and WJ-MSCs groups for up to 16 weeks following transplantation surgery.(B) Measurement of the isometric tetanic force at 16 weeks postoperatively.(C) Microscopic examination of toluidine blue-stained distal sciatic nerve ends at 16 weeks postoperatively showing the histological structures of nerve fiber, axon, and myelin.Scale bar = 20 μm.(D) Total axon count is shown.The mean ± SEM values of the control and WJ-MSCs groups are compared (*p < 0.05, **p < 0.01, Student's t-test).

Figure 2 .
Figure 2. Analysis of the expression of macrophage markers.(A) RT-qPCR analysis of CD206 and IL10 in the control and WJ-MSCs groups.Expression levels were normalized against GAPDH expression.(B) Immunofluorescence for CD206 and CD68 in cross-sections at 3, 7, and 14 days post-implantation as indicated.Scale bar = 500 μm (*p < 0.05, Student's t-test).

Figure 3 .
Figure 3. Analysis of the expression of angiogenesis and neurotrophic factors.(A) RT-qPCR analysis of NGF, BDNF, and VEGF in the control and WJ-MSCs groups.Expression levels were normalized against GAPDH expression.(B) Immunofluorescence for NGF, BDNF, and VEGF in cross-sections at 3, 7, and 14 days posttransplantation as indicated.Scale bar = 500 μm (*p < 0.05, Student's t-test).

Figure 4 .
Figure 4. Analysis of the expression of SC markers.(A) RT-qPCR analysis of S100b and MBP in the control and WJ-MSCs groups.Expression levels were normalized against GAPDH expression.(B) Immunofluorescence for S100β in cross-sections at 3, 7, and 14 days post-transplantation as indicated.Scale bar = 500 μm (*p < 0.05, Student's t-test).

Figure 5 .
Figure 5. Analysis of double-staining of S100β and human nuclei.Immunofluorescence co-staining of human nuclei and S100β in cross-sections of the control and WJ-MSCs groups at 3 and 7 days.Scale bar = 200 μm.